Urokinase-type plasminogen activator (uPA) has been identified as the initiator of a major amplified cascade of extracellular proteolysis. This cascade, when regulated, is vital to certain normal physiological processes but, when dysregulated, is strongly linked to pathological processes, such as cell invasion and metastasis in cancer. Dano/ et al., Adv. Cancer Res., 44:139-266 (1985). Cells express uPA as an inactive form, pro-uPA or single-chain uPA, which then binds to its receptor, uPAR. This binding event is necessary for activation to two-chain uPA. Ellis et al., J. Biol. Chem., 264:2185-88 (1989). The amino acid sequence for human pro-uPA is as follows (SEQ ID NO:1):
Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly 1 10 Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys 20 30 Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile 40
The sequence of amino acids of pro-uPA are represented above by their standard three-letter abbreviations as follows:
______________________________________ Amino Acid Three-letter Symbol ______________________________________ Alanine Ala Arginine Arg Asparagine Asn Aspartic acid Asp Cysteine Cys Glutamine Gln Glutamic acid Glu Glycine Gly Histidine His Isoleucine Ile Leucine Leu Lysine Lys Methionine Met Phenylalanine Phe Proline Pro Serine Ser Threonine Thr Tryptophan Trp Tyrosine Tyr Valine Val ______________________________________
The structure of pro-uPA is shown in FIG. 1.
uPA is a three-domain protein comprising (1) an N-terminal epidermal growth factor-like domain, (2) a kringle domain, and (3) a C-terminal serine protease domain. The receptor for pro-uPA (uPAR) is a multi-domain protein anchored by a glycolipid to the cell membrane, thus ensuring that activation of uPA is a pericellular event. Behrendt et al., Biol. Chem. Hoppe-Seyler, 376:269-79 (1995). uPA activity is confined to the cell surface by plasminogen activator inhibitors (PAI-1 and PAI-2), which bind to and inactivate the bound uPA. This tight control of uPA activity is necessary because uPA acts upon a substrate, plasminogen, that is present at a high concentration in plasma. Robbins, Meth. Enzymol., 19:184-99 (1970). The product of uPA's action upon plasminogen, plasmin, is a powerful broad spectrum protease that not only degrades extracellular matrix proteins directly, but also activates the latent forms of other proteases, including several metalloproteases. Werb et al., N. Eng. J. Med., 296:1017-1023 (1977); Mignatti et al., Cell, 47:487-98 (1986); He et al., Proc. Natl. Acad. Sci. USA, 86:2632-36 (1989); and Matrisian, Bioessays, 14:455-63 (1992).
In tumor biology, the link between extracellular proteolysis and angiogenesis is clearly evident. Break-up and dissolution of existing extracellular matrix is necessary in order to create new space for blood vessels to grow into. The processes of proteolysis and angiogenesis are highly coordinated. For example, two pre-eminent angiogenic growth factors, basic fibroblast growth factor and vascular endothelial growth factor markedly up-regulate the production of uPA. (Montesano et al., Proc. Natl. Acad. Sci. USA, 83:7297-7301 (1986); Pepper et al., Biochem. Biophys. Res. Comm., 181:902-906 (1991)) and the expression of uPAR by endothelial cells (Mignatti et al., J. Cell Biol., 113:1193-1201 (1991); Mandriota et al., J. Biol. Chem., 270:9709-9716 (1995)). Thus uPA/uPAR has emerged as a new target for developing an anti-metastatic/anti-angiogenic therapy for cancer, where most studies have been conducted (Fazioli et al., Trends Pharmacological Sci., 15:25-29 (1994).
However, the uPA/uPAR interaction goes far beyond localizing proteolysis at the cell surface. Independent of all proteolytic effects, the mere occupation of uPAR by uPA induces, by indirect means, signal transduction events leading to one or more of the following effects: mitogenesis (Rabbani et al., J Biol. Chem., 267:14151-56 (1992)); expression of the c-fos gene (Dumler et al., FEBS Lett. 322:37-40 (1994)); cysteine- and metalloprotease expression by macrophages (Rao et al., J. Clin. Invest. 96:465-74 (1995)): transfer of mechanical force leading to increased cytoskeletal stiffness (Wang et al., Am. J. Physiol. 268:C1062-C1066 (1995)); endothelial cell migration (Odekon et al., J. Cellul. Physiol., 150:258-63 (1992)); endothelial cell morphogenesis into tubular structures (Schnaper et al., J. Cellul. Physiol. 165:107-118 (1995)); and endothelial cell deformability and motility (Lu et al., FEBS Lett. 380:21-24 (1996)). All of these phenomena are blocked by blocking the access of uPA to uPAR. An antagonist of uPAR that prevented the binding of uPA would thus interfere with proteolytic activity by preempting uPA activation and, further, would greatly diminish uPAR's capacity for signal transduction.
The anti-angiogenic effects accompanying uPAR antagonism (Min et al., Cancer Res., 56:2428-33 (1996)) should allow a uPAR antagonist to play a role in other diseases characterized by inappropriate angiogenesis, e.g. ocular angiogenesis leading to blindness. Furthermore, it is likely that a uPAR antagonist would also play a therapeutic role in inflammatory diseases, for example, rheumatoid arthritis. (Ronday et al., Br. J. Rheum., 35:416-423 (1996).
One approach to drug therapy is to target uPA itself at its catalytic serine protease domain. Yang et al., Fibrinolysis, 6 (Suppl. 1):31-34, (1992). Amiloride (Vassalli et al., FEBS Lett., 214:187-191 (1987); and Kellen et al., Anticancer Res. 8:1373-76 (1988)) and p-aminobenzamidine (Geratz et al., Thrombosis Res. 24:73-83 (1981); and Billstrom et al., Int. J. Cancer, 61:542-47 (1995)) are competitive inhibitors of this site and have anti-metastatic activity in vivo. Selective inhibition of uPA as compared with other serine proteases, was evident in phenylguanidines (Yang et al., J. Med. Chem., 33:2956-61 (1990)) and, even more so, in benzob!thiophene-2-carboxamidines (Bridges, Bioorganic & Medicinal Chemistry, 1:403-410 (1993); Towle et al., Cancer Res., 53:2553-59 (1993); and Rabbani et al., Int. J. Cancer, 63:840-45 (1995)).
Towards defining the binding epitope for the uPA-uPAR interaction, it was first shown that the amino terminal fragment of uPA (residues 1-135) that lacked the serine protease domain, sufficed for high affinity, sub-nanomolar binding. (Stoppelli et al., Proc. Natl. Acad. Sci. USA 82:4939-43 (1985). Further work showed that the growth factor domain alone (residues 1-48) conferred this binding. (Robbiati et al., Fibrinolysis, 4:53-60 (1990); and Stratton-Thomas et al., Protein Engineering 8:463-70 (1995.)) Dano/ et al., in WO 90/12091 published 18 Oct., 1990, discloses that the binding of uPA to uPAR could be prevented by administering a substance comprising a sequence identical or substantially identical to a uPAR binding site of uPA amino residues 12-32. WO 94/28145, by Rosenberg and Stratton-Thomas, Dec. 8, 1994, discloses the preparation and use of de-fucosylated HuPA.sub.1-48 that prevents uPA binding to uPAR.
Earlier studies with peptide fragments within the growth factor domain had showed that residues 20-30 conferred the specificity of binding, but that residues 13-19 were needed in addition for residues 20-30 to attain the proper binding conformation. Specifically, the peptide Ala.sup.19 !uPA-(12-32) (SEQ ID NO:2, which contains two cysteines (the third cysteine being replaced by Ala to avoid undesired disulfide bond formations), in its open chain form prevented uPA binding to uPAR with an IC.sub.50 of 100 nM. In its oxidized cyclic form, having an intrachain disulfide bond between Cys.sup.13 and Cys.sup.31, the peptide prevented binding with an IC.sub.50 of 40 nM. It was proposed that residues 13-19 might act indirectly to provide a scaffold that would help residues 20-30 attain the correct binding conformation. Appella et al., J. Biol. Chem., 262:4437-40 (1987).
These results were partially confirmed when it was reported that, while the linear peptide 20-30 (SEQ ID NO:3) inhibited the binding of uPA to uPAR with an IC.sub.50 of 1,000 nM, the longer peptide 17-34 (SEQ ID NO:4) was significantly more potent, having an IC.sub.50 of 100 nM. It was also shown that the corresponding longer peptide (17-34) derived from the mouse sequence inhibited spontaneous metastasis of a murine Lewis Lung carcinoma in mice, whereas the corresponding linear shorter peptide (20-30) (SEQ ID NO:3) had no effect. Kobayashi et al., Int. J. Cancer, 57:727-33 (1994). WO 94/28014 by Rosenberg and Doyle, Dec. 8, 1994 discloses the preparation and use of 25 random peptides displayed on bacteriophage which competed with the N-terminal fragment of uPA for binding to uPAR with IC.sub.50 values of 15 nM to &gt;50 .mu.M.
Most recently, Magdolen et al., "Systematic Mutational Analysis of the Receptor-binding Region of the Human Urokinase-type Plasminogen Activator", Eur. J. Biochem., 237:743-51 (1996), describes alanine-scanning mutagenesis of the binding loop of the amino-terminal fragment of uPA with the finding that Asn22, Lys23, Tyr24, Phe25, Ile28 and Trp30 are important side chains that should be kept. Further, Magdolen et al., citing Hansen et al., Biochemistry, 33:4847-64 (1994), disclose that the region between Thr18 and Asn32 consists of a flexible, seven-residue omega loop that is forced into a ring-like structure. Although Cys19 and Cys31 are in close proximity to each other (0.61 nm), they do not form a disulfide bond with each other. Instead Cys19 forms a disulfide bond with Cys11, and Cys31 forms a bond with Cys13. See FIG. 2 (SEQ ID NO:5). Accordingly, the uPAR binding site on uPA does not form a simple, small ring structure.
Some scientists have explored the possibility of cyclizing the one or more of the growth factor domains of peptide analogues to increase their competitive binding activity, but not with any great success without at least adding some other constraining-type modifications of the structure. For example, in Chamberlin et al., J. Biol. Chem., 270:21062-21067 (1995), peptides constrained by the introduction of an intramolecular disulfide bond also required the substitution of another entity for proline in the peptide loop to achieve any significant activity. Lougheed et al., Protein Sci., 4:773-80 (1995) found that peptides from the fifth EGF-like domain of thrombomodulin had very weak biological activity that increased marginally (two-fold) by cyclization. The additional presence of a "tail" of amino acids and the deletion of one of the amino acids were both found necessary and, even then, the best peptide was only weakly active (text of micromolar range). Thus, cyclization per se conferred no significant activity. Further, others working in the thrombomodulin field have found that the number of crossing disulfide bonds in the fifth EGF-like domain is inversely, rather than directly, related to inhibitory potency. Hunter et al., Protein Sci., 4:2129-37 (1995).
It has now been found by the present inventors that novel cyclic structures derived from the peptide fragment 20-30, in which residue 20 is covalently bonded to residue 30, do exhibit the ability to bind to uPAR and are also antagonists of the binding of uPA to uPAR. These peptides are shorter than either Ala.sup.19 !uPA-(12-32) (Appella et al., supra.) or uPA17-34 (Kobayashi et al., supra.), but bind almost as effectively. In contradiction of what was hitherto thought, it has been discovered that the eight amino acids N-terminal to Val20 in Ala.sup.19 !uPA-(12-32) and the four amino acids CO-terminal to Trp30 in uPA17-34 are not necessary for high binding affinity. While not wishing to be bound by any particular theory, it now appears that the minimal binding epitope in urokinase-type plasminogen activator, which is needed for binding to its receptor, is a loop of only eleven amino acids.